Counterdefense Against RNA Silencing By Tomato Yellow Leaf Curl China Virus And Molecular Variation Of Tomato Yellow Leaf Curl Virus

The Geminiviridae is a large family of plant viruses with circular, single-stranded DNA genomes that cause devastating diseases of economically important crops world-wide. To get a better insight into the pathogenesis and epidemics of geminiviruses, the mechanism of geminivirus counterdefense against RNA silencing as well as the molecular variation of geminiviruses were studied.1 Characterization of small interfering RNAs derived from Tomato yellow leaf curl China virus and its associated betasatelliteWe profiled Tomato yellow leaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB)-derived small RNAs (V-sRNAs and S-sRNAs) using Solexa-based deep sequencing to evaluate the role of betasatellites in V-sRNA modulation. Both sense and anti-sense V-sRNAs and S-sRNAs accumulated preferentially as 22,21 and 24 nucleotide species in infected Solanum lycopersicum and Nicotiana benthamiana plants. High resolution mapping of V-sRNA and S-sRNA revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV andβC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5'terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3'terminal of AC1, indicating the important role ofβC1 in V-sRNA modulation.βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. Our results suggest that increased viral transcript levels might result in aberrant RDR substrates resulting in dsRNA and secondary siRNA production.2 Suppression of methylation-directed transcriptional gene silencing by TYLCCNBβC1 The study focused on the suppression of DNA methylation and transcriptional gene silencing (TGS) by TYLCCNV and its associated TYLCCNB. Using bisulfite sequencing, we found that cytosine residues in the TYLCCNV genome are subject to DNA methylation in infected N. benthamiana plants, and that methylation appears to be enriched at sequences within or near promoters, including the intergenic region (IR) that contains divergent promoters flanking the origin of replication, the downstream complementary sense promoters within the AC1/AL1 coding region that drive expression of AC2/AL2 and AC3/AL3, and sequences within the AV2 and AV1 reading frames that include the AV1/CP (coat protein) promoter. Following co-inoculation of TYLCCNB with TYLCCNV, methylation of the helper virus is substantially reduced from～5.4% to～1.25% in infected plants. Infectivity tests show that TYLCCNB can complement Beet curly top virus (BCTV) L2- mutants and prevents host recovery, and reduce the methylation of BCTV IR by about 15%. Using a methylation-sensitive single-nucleotide extension assay, we show that TYLCCNB cause global reduction of cytosine methylation in host genomic DNA by approximately 2.5 fold. Co-inoculation of TYLCCNB with either TYLCCNV or BCTV L2- can reverse TGS of a green fluorescent protein (GFP) transgene in N. benthamiana line 16-TGS, while unlike BCTV, TYLCCNV by itself is ineffective on TGS suppression. Over-expression ofβC1 suggested thatβC1 can reverse TGS of GFP transgene in N. benthamiana line 16-TGS, and cause ectopic expression of endogenous F-box loci silenced by methylation as well as global reduction in cytosine methylation of Arabidopsis genomic DNA. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed thatβC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. In vitro SAHH activity assays with purified proteins confirm that the interaction is direct, and show that physical interaction results in substantial reduction of SAHH activity by about 80%. ThatβC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product. S-adenosyl methionine, may be a common viral strategy for methylation interference. 3 Molecular variation of the natural population of Tomato yellow leaf curl virusTo get a better insight into the evolutionary trajectory of Tomato yellow leaf curl virus (TYLCV) in China,26 TYLCV isolates were collected from Shanghai during 2006-2010. Sequence analysis of 122 full length TYLCV sequences obtained by rolling circle amplification (RCA) showed that they shared 98.5%-100% nucleotide sequence similarity. The mean genomic substitution rate was estimated to be 1.69×10-3 nucleotide substitutions per site per year (subs/site/year), which was comparable to that reported for plant RNA viruses. Estimation of the intergenic region and individual ORFs showed that the intergenic region was more variable than the other ORFs, with a mean substitution rate of 4.81×10-3 subs/site/year. Further analysis of the sequences indicated that the high mutation rate was mainly due to nucleotide substitution, with nucleotide substitution biases for C→T, T→C, G→A and G→T. Our results further support the notion that geminiviruses evolve as rapidly as many RNA viruses.4 Molecular variation of TYLCV in Bemisia tabaciTo get a better insight into the variation of TYLCV in its transmission vector, invasive Q biotype and indigeneous ZHJ2 biotype whiteflies were fed on S. lycopersicum plants experimentally infected with infectious clone of TYLCV. Full length TYLCV sequences were cloned from viruliferous Q and ZHJ2 biotype whiteflies after 12 h and 15 day post acquisition using RCA. Our results demonstrate that the populations of TYLCV were genetically heterogenous and that rapid mutation occurred in viruliferous whiteflies. We also showed that the nucleotide diversity of the TYLCV populations from viruliferous whiteflies was higher than that from experimentally infected S. lycopersicum plants, with a mean substitution rate of 4.08×10-3 and 3.45×10-3 in Q and ZHJ2 biotype whiteflies, respectively. Genetic differentiation analysis showed significant differences among the populations from viruliferous whiteflies and infected S. lycopersicum plants. Further analyses showed that the high mutation rate of TYLCV in viruliferous whiteflies was mainly due to the frequent mutation of nucleotide at several positions, with nucleotide biases for C→T, G→A, A→G and T→G. In summary, we highlight the important role of B. labaci in TYLCV evolution.